Bone marrow-derived hematopoietic stem cells (HSCs) have been shown to target sites of neovascularization in the retina. These HSCs have vasculo- and neurotrophic rescue in mouse models of retinal degeneration, and have enormous potential for clinical use as therapeutics for treating retinal degenerative diseases. However, the mechanism by which these cells exert their trophic activity remains unknown. Therefore, in an effort to characterize the activity of these cells, significant improvements need to be made in their extraction/identification. This research will develop a cell-based positive selection assay utilizing small molecules. We will accomplish this through three specific aims Specific Aim 1: Develop a high through-put screening assay to test for small molecules that preferentially bind to CD44HI-expressing cells. Using a pre-competitive cell based ELISA-like assay, we will screen a non-peptidic library for small molecules that bind efficiently to CD44HI-expressing cells. A colorimetric assay will be used to assay the small molecules ability to inhibit binding to bone marrow derived cells by an antibody to CD44. Specific Aim 2: Optimize the binding efficiency of the small molecule through chemical modifications. Enlisting the solution phase methodology used for the library synthesis, secondary libraries will be prepared based on systematic modifications of the validated leads identified in the high through-put assay and optimized for binding affinity inhibiting antibody binding with CD44Hi-expressing cells. Specific Aim 3: Utilize identified small molecules to positively sort out CD44Hi-expressing cells and assess trophic/ biological activity of these cells in animal models of retinal neuronal/vascular degeneration. By incubating whole bone marrow with the identified small molecule(s) and then sorting for cells bound to the small molecule, we can analyze these cells to see if they are CD44Hi-expressing and/or possess trophic activity. This will be accomplished by injecting this subpopulation of cells intravitreally into mice with retinal degeneration and observing rescue of vascular and neuronal degeneration as previously described. In retinal diseases such as diabetic retinopathy, and age-related macular degeneration, the leading causes of vision loss in adults, abnormal blood vessel growth occurs. This research will contribute to the development of therapeutic drugs aimed at treating retinal diseases caused by blood vessel malformation that can result in blindness.